A Role for sFRP5 in Adipocyte Biology and Obesity by Tyler C. Prestwich A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Cellular and Molecular Biology) in The University of Michigan 2008 Doctoral Committee: Professor Ormond A. MacDougald, Chair Professor Charles Burant Professor Eric R. Fearon Associate Professor Kenneth M. Cadigan Assistant Professor Peter C. Lucas Tyler C. Prestwich 2008 Table of Contents List of Figures iv Chapter I. Wnt/β-catenin Signaling in Adipogenesis and Metabolism 1 Summary 1 Introduction 1 Wnt Signaling Regulates Adipogenesis In Vitro and In Vivo 3 Wnt/β-catenin Signaling and Human Metabolic Disorders 6 Recent Advances in Wnt/β-catenin-Mediated Effects on Adipogenesis 7 Regulation of Mesenchymal Cell Fate 7 β-catenin as a Central Mechanism for Inhibiting Adipogenesis 9 Conclusions 10 II. A Role for sFRP5 in Adipocyte Biology and Obesity 18 Abstract 18 Introduction 19 Materials and Methods 21 Results 30 Expression and Localization of sFRP5 In Vitro and In Vivo 30 sFRP5 is Associated with Increasing Adiposity and Adipocyte Size 31 sFRP5Q27stop Mice Resist Diet-Induced Obesity and Display a Reduction in Fat Mass 32 sFRP5 Regulates Adipocyte Size During Obesity in a Tissue Autonomous Manner 34 Relative Expression of Adipocyte and Vasculature Markers in sFRP5Q27stop Mice 36 Metabolic Phenotype of Mice Harboring the sFRP5Q27stop Mutation 38 Integrin Signaling may Mediate sFRP5-Regulated Adipocyte Clustering 39 Discussion 42 III. Expanding the Scope of sFRP5 Function 61 Introduction 61 Materials and Methods 62 ii Results 64 Temperature Dependent Effects on Food Intake in sFRP5Q27stop Mice 64 µCT Analysis of Cortical and Trabecular Bone in sFRP5Q27stop Mice 65 Microarray Analysis of G-WAT from sFRP5Q27stop mice 66 Discussion 68 IV. Future Directions 78 Summary of Results 78 What Regulates sFRP5 Expression in Adipogenesis and Obesity? 82 Which Signaling Pathways Mediate sFRP5-Dependent Effects? 83 Which Cell Types are Targeted by sFRP5? 86 iii List of Figures Figure 1.1 Wnt/β-catenin Signaling is a Central Pathway Regulating Adipogenesis 11 2.1 Expression and Localization of sFRP5 In Vitro and In Vivo 48 2.2 sFRP5 is Associated with Increasing Adiposity and Adipocyte Size 49 2.3 sFRP5Q27stop Mice Resist Diet-Induced Obesity and Display a Reduction in Fat Mass 50 2.4 sFRP5 Regulates Adipocyte Size During Obesity in a Tissue Autonomous Manner 51 2.5 Relative Expression of Adipocyte and Vasculature Markers in sFRP5Q27stop Mice 52 2.6 Metabolic Phenotype of Mice Harboring the sFRP5Q27stop Mutation 53 2.7 Integrin Signaling may Mediate sFRP5-Regulated Adipocyte Clustering 54 2.8 sFRP5 Regulates Adipocyte Growth and Preadipocyte Recruitment 55 3.1 Temperature Dependent Effects on Food Intake in sFRP5Q27stop Mice 72 3.2 µCT Analysis of Cortical and Trabecular Bone in sFRP5Q27stop Mice 73 3.3 Ingenuity Pathway Analysis of Cytochrome C Oxidase Subunit Upregulation in sFRP5Q27stop mice 74 3.4 Ingenuity Pathway Analysis of NADH2 Dehydrogenase and ATP synthase Subunit Upregulation in sFRP5Q27stop Mice 75 iv Chapter I Wnt/β-catenin Signaling in Adipogenesis and Metabolism Summary Adipocyte differentiation consists of a complex series of events in which scores of cellular and extracellular factors interact to transform a fibroblast-like preadipocyte into a mature, lipid-filled adipocyte. Many of the pathways influencing this process have been identified using well-characterized preadipocyte culture systems and have subsequently been confirmed in animal models. Research conducted over the last decade has established the Wnt/β- catenin signaling pathway as an important regulator of adipocyte differentiation. While initial reports implicated activators of Wnt/β-catenin signaling as potent inhibitors of adipogenesis, recent investigations of mesenchymal cell fate, obesity, and type 2 diabetes highlight significant additional roles for Wnt signaling in metabolism and adipocyte biology. Introduction Adipogenesis is the process by which mesenchymal precursor cells differentiate into adipocytes, which store lipid and serve as central regulators of metabolism [1-3]. Identifying key factors that control adipocyte differentiation and metabolism is vital to understanding adipose tissue biology and pathology. The 1 transcriptional cascade controlling adipogenesis has been well characterized over the past two decades and mechanisms by which master adipocyte regulators act are now beginning to be fully elucidated. Peroxisome proliferator- activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) are the chief regulators thought to coordinately direct the adipogenic program. PPARγ is both necessary and sufficient for preadipocyte differentiation [1], while C/EBPα appears to be important for the acquisition of insulin sensitivity in adipocytes [4]. The current state of research on these important transcriptional regulators has been recently reviewed elsewhere [2,3]. Transcription factors that control the cascade of events leading to a fully differentiated adipocyte act downstream of complex signaling pathways that integrate signals from the surrounding microenvironment. Over the past several years, the field of adipogenesis has seen an upsurge in the number of reports implicating locally secreted or circulating extracellular factors as regulators of preadipocyte differentiation [3]. One of the extracellular signaling pathways now known to affect adipogenesis is the Wnt pathway. Wnts are an evolutionarily conserved family of secreted lipidated glycoproteins with well-established roles in cellular proliferation, differentiation, and polarity during embryogenesis [5,6]. More recently, Wnt signaling has been shown to modulate additional developmental and physiological processes, including aspects of adipocyte biology [7-11]. In this review, we provide an overview of the research revealing a principal role for Wnt signaling in adipogenesis. We present a brief chronology of the studies demonstrating Wnt inhibition of adipocyte differentiation in vitro and in 2 vivo, culminating with the recent linkages of Wnt pathway members to human diseases including obesity and type 2 diabetes. Finally, we examine the latest reports providing mechanistic insight into how Wnt signaling functions to block adipogenesis and regulate mesenchymal cell fate. Wnt Signaling Regulates Adipogenesis In Vitro and In Vivo: Wnts are secreted proteins that act though autocrine and paracrine mechanisms to influence the development of many cell types [5,6]. Although Wnts can inhibit preadipocyte differentiation through both β-catenin-dependent and -independent mechanisms [12], current genetic evidence supports β-catenin as a particularly crucial regulator of adipogenesis [13]. In the canonical Wnt signaling pathway, β-catenin plays a central role as a transcriptional coactivator. Upon binding of Wnt ligands to frizzled receptors and low density lipoprotein receptor-related protein (LRP) coreceptors, cytoplasmic β-catenin is hypophosphorylated, stabilized, and translocated into the nucleus where it binds to and coactivates members of the T-cell factor/lymphoid-enhancing factor (TCF/LEF) family of transcription factors to direct target gene expression (Fig. 1.1). In Vitro Studies utilizing preadipocyte lines originally demonstrated that ectopic expression of Wnt1, an activator of Wnt/β-catenin signaling, potently represses adipogenesis (Fig. 1.1) [7,11]. Similarly, pharmacological agents that activate 3 canonical Wnt signaling and stabilize free cytosolic β-catenin also block preadipocyte differentiation [7,8]. Conversely, inhibition of Wnt signaling in preadipocytes stimulates differentiation [7,8,14-16], suggesting that preadipocytes produce endogenous Wnts that strongly repress adipogenesis. One endogenous factor is Wnt10b, expression of which is high in dividing and confluent preadipocytes and is rapidly downregulated in response to elevated cAMP during induction of differentiation [7,8]. Furthermore, constitutive expression of Wnt10b stabilizes free cytosolic β-catenin and inhibits adipogenesis (Fig. 1.1) [7]. While considerable evidence suggests that Wnt10b is a prominent extracellular regulator of adipogenesis, other Wnt ligands are also expressed and likely contribute to the process. For example, Wnt6 and Wnt10a have been identified as endogenous regulators of brown adipocyte development [17,18]. Additionally, Wnt5b is transiently induced during adipogenesis and acts through an unknown mechanism to destabilize β-catenin and promote differentiation [19,20], indicating that preadipocytes integrate inputs from a variety of competing Wnt signals (Fig. 1.1). One of the mechanisms by which Wnt/β-catenin signaling inhibits adipogenesis is thought to involve dysregulated expression of cyclin dependent kinase inhibitors, p21 and p27 [21]. Adipogenesis is regulated not only by expression of specific Wnt ligands, but also by expression of factors that inhibit the Wnt/β-catenin pathway. For example, Li et al. recently reported that a nuclear β-catenin antagonist, chibby (Cby), is expressed in adipose tissue and is induced during differentiation of 3T3- L1 preadipocytes (Fig. 1.1) [14]. Cby binds the C-terminal portion of β-catenin 4 and blocks interaction with TCF/LEF transcription factors, thus repressing β- catenin-mediated transcriptional activation [22]. Ectopic expression of Cby in 3T3-L1 cells induces spontaneous differentiation into mature adipocytes, while depletion of Cby stimulates β-catenin